Oligonucleotide analog and method for treating flavivirus infections

ABSTRACT

A method of inhibiting replication of a flavivirus in animal cells, and an oligonucleotide compound for use in the method are disclosed. The oligonucleotide analog (i) has a nuclease-resistant backbone, (ii) is capable of uptake by the cells, (iii) contains between 8-40 nucleotide bases, and (iv) has a sequence of at least 8 bases complementary to a region of the virus&#39; positive strand RNA genome that includes at least a portion of SEQ ID NOS:1-4. Exposure of cells infected with a flavivirus to the analog is effective to form within the cells, a heteroduplex structure composed of the virus ssRNA and the oligonucleotide, characterized by a T m  of dissociation of at least 45° C., and having disrupted base pairing between the virus&#39;s 5′ and 3′ cyclization sequences.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/848,873 filed Aug. 2, 2010 (pending); which is a divisional of U.S.application Ser. No. 10/913,996 filed Aug. 5, 2004 (U.S. Pat. No.7,807,801); which claims priority to U.S. Provisional Application No.60/493,043 filed Aug. 5, 2003 and U.S. Provisional Application No.60/512,003 filed Oct. 16, 2003, all of which are incorporated herein byreference in their entireties.

FIELD OF THE INVENTION

This invention relates to an oligonucleotide analog for use in treatinga flavivirus infection in animals, to an antiviral method employing theanalog, and to a method for monitoring binding of the analog to a viralgenome target site.

REFERENCES

The following references are related to the background or to methods orprotocols that may be employed in the invention.

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Coats (1969). “Synthesis of carbonate    analogs of dinucleosides. 3′-Thymidinyl 5′-thymidinyl carbonate,    3′-thymidinyl 5′-(5-fluoro-2′-deoxyuridinyl) carbonate, and    3′-(5-fluoro-2′-deoxyuridinyl) 5′-thymidinyl carbonate.” J Med Chem    12(1): 154-7.-   Mongkolsapaya, J., W. Dejnirattisai, et al. (2003). “Original    antigenic sin and apoptosis in the pathogenesis of dengue    hemorrhagic fever.” Nat Med 9(7): 921-927.-   Morrey, J. D., D. F. Smee, et al. (2002). “Identification of active    antiviral compounds against a New York isolate of West Nile virus.”    Antiviral Res 55(1): 107-16.-   Moulton, H. M., M. H. Nelson, et al. (2004). “Cellular uptake of    antisense morpholino oligomers conjugated to arginine-rich    peptides.” Bioconjug Chem 15(2): 290-9.-   Proutski, V., E. A. Gould, et al. (1997). “Secondary structure of    the 3′ untranslated region of flaviviruses: similarities and    differences.” Nucleic Acids Res 25(6): 1194-202.-   Scherret, J. H., M. Poidinger, et al. (2001). “The relationships    between West Nile and Kunjin viruses.” Emerg Infect Dis 7(4):    697-705.-   Ternovoi, V. A., G. P. Kurzhukov, et al. (2003). “Tick-borne    encephalitis with hemorrhagic syndrome, novosibirsk region, Russia,    1999.” Emerg Infect Dis 9(6): 743-6.-   Toulme, J. J., R. L. Tinevez, et al. (1996). “Targeting RNA    structures by antisense oligonucleotides.” Biochimie 78(7): 663-73.-   You, S., B. Falgout, et al. (2001). “In vitro RNA synthesis from    exogenous dengue viral RNA templates requires long range    interactions between 5′- and 3′-terminal regions that influence RNA    structure.” J Biol Chem 276(19): 15581-91.-   Zeng, L., B. Falgout, et al. (1998). “Identification of specific    nucleotide sequences within the conserved 3′-SL in the dengue type 2    virus genome required for replication.” J Virol 72(9): 7510-22.

BACKGROUND OF THE INVENTION

The family Flaviviridae is a group of single, positive-stranded RNAviruses with a genome size from 9-15 kb. They are enveloped viruses ofapproximately 40-50 nm. Within the Flaviviridae family is the flavivirusgenus which includes the prototype yellow fever virus (YFV), the fourserotypes of dengue virus (DEN-1, DEN-2, DEN-3, and DEN-4), Japaneseencephalitis virus (JEV), Murray Valley encephalitis virus (MVEV),Kunjin virus (KUN), St. Louis encephalitis virus (SLEV), West Nile virus(WNV), Tick-borne encephalitis virus (TBEV), and about 70 other diseasecausing viruses.

Most flavivirus infections are treated with supportive measures such asanti-pyretics to keep fever down, fluids, antibiotics for secondarybacterial infection, respiratory support as necessary, etc. The use ofribavirin has significant antiviral chemotherapeutic activity against anumber of RNA viruses and has been proven to be effective in thetreatment of Infuenza virus, Respiratory syncytial virus, Lassa fevervirus, and Hanta virus infections. Ribavirin in combination with variousinterferon drugs is used to treat Hepatitis C virus infections. However,the in vitro and in vivo activity of ribavirin against flaviviruses suchas dengue and yellow fever is very weak (Leyssen, De Clercq et al.2000).

Despite four decades of research effort, safe and effective vaccinesagainst most flaviviruses such as dengue are still not available.Although effective vaccines against yellow fever virus and Japaneseencephalitis virus exist, these viruses still cause significant diseaseworldwide. Efforts to develop an effective dengue vaccine arecomplicated by the epidemiology of the virus. Although immunity to anygiven dengue serotype induces lifelong immunity for that particularserotype, a second infection with a different serotype can induce denguehemorrhagic fever and dengue shock syndrome (DHF/DSS), the severe formsof dengue infection with associated mortality over 20% (Mongkolsapaya,Dejnirattisai et al. 2003).

In view of the severity of the diseases associated with flavivirusinfection and their pervasiveness in animals and especially man, thereis a need for therapeutic compounds and methods for treating a hostinfected with a flavivirus.

SUMMARY OF THE INVENTION

The invention includes, in one aspect, a method of inhibitingreplication of a flavivirus in animal cells, including mammalian andavian cells. In practicing the method, cells are exposed to anoligonucleotide analog (i) having a nuclease-resistant backbone, (ii)capable of uptake by the cells, (iii) containing between 8-40 nucleotidebases, and (iv) having a sequence of at least 8 bases complementary to aregion of the virus' positive strand RNA genome that includes at least aportion of one of sequences identified as SEQ ID NOS:1-4, each sequencerepresenting the viral genome's 5′ or 3′ cyclization sequence in one oftwo broad class of flaviviruses. In a preferred embodiment, theoligonucleotide analog has a sequence that is complementary to at leasta portion of the 3′ cyclization sequence of SEQ ID NOS: 3 or 4 in aviral genome's positive strand. Exposure to the compound is effective toform within the cells, a heteroduplex structure composed of the virusssRNA and the oligonucleotide, characterized by a T_(m) of dissociationof at least 45° C., and having disrupted base pairing between the virus'5′ and 3′ cyclization sequences, as evidenced by inhibition of viralreplication in the cells.

In one embodiment, the analog is complementary to all or a portion ofone of the sequences of SEQ ID NOS:1-4. In another embodiment, theanalog contains a sequence that is capable of forming a heteroduplexstructure with a viral sequence that includes of a portion of thegenome's 5′ cyclization sequence and a complementary portion of thegenome's 3′ cyclization sequence.

For use in inhibiting replication of any of St. Louis encephalitisvirus, Murray Valley encephalitis virus, West Nile virus, Kunjin virus,Japanese encephalitis virus, Yellow fever virus, Dengue virus—Types 1,2, 3, and 4, or West Nile virus, the oligonucleotide analog to which thecells are exposed has a sequence of at least 8 bases complementary to aregion of the virus' positive strand RNA genome that includes at least aportion of SEQ ID NOS:1 or 3, and preferably SEQ ID NO:3. In anotherembodiment, the oligonucleotide analog contains a sequence capable offorming a heteroduplex structure with a sequence that includes of aportion of the genome's 5′ cyclization sequence identified as SEQ IDNO:1 and a complementary portion of the genome's 3′ cyclization sequenceidentified as SEQ ID NO:3.

For use in inhibiting replication of any of Tick borne encephalitisvirus, Powassen virus, Louping III virus, Kyasanur Forest disease virus,and Alkhurma virus, the oligonucleotide analog to which the cells areexposed has a sequence of at least 8 bases complementary to a region ofthe virus' positive strand RNA genome that includes at least a portionof SEQ ID NOS: 2 or 4, and preferably SEQ ID NO:4. In anotherembodiment, the oligonucleotide analog contains a sequence capable offorming a heteroduplex structure with a sequence that includes a portionof the genome's 5′ cyclization sequence identified as SEQ ID NO:2 and acomplementary portion of the genome's 3′ cyclization sequence identifiedas SEQ ID NO:4.

A preferred oligonucleotide analog has an uncharged, or substantiallyuncharged, backbone, such as one of the structures shown in FIGS. 2A-2G,and is preferably composed of 8-25 morpholino subunits linked by asubstantially uncharged, phosphorous-containing backbone, such as thestructures shown in FIGS. 3A-3D. One preferred analog has the structureshown in FIG. 3B, where X is NR₂, where R is H or CH₃, and Y and Z areeach 0.

For treating a flavivirus infection in an animal subject, including ahuman subject, the infected cells may be exposed to the oligonucleotideanalog by parenteral or oral administration to the infected subject. Themethod may further include monitoring a body fluid for the appearance ofa heteroduplex composed of the oligonucleotide analog and acomplementary portion of the viral genome.

In another aspect, the invention includes an oligonucleotide analog foruse in inhibiting replication of a flavivirus in animal cells. Theanalog is characterized by: (i) a nuclease-resistant backbone, (ii)capable of uptake by animal cells, (iii) containing between 8-40nucleotide bases; (iv) having a sequence of at least 8 basescomplementary to a region of the flavivirus's positive strand RNA genomethat includes at least a portion of one of the viral genome sequencesidentified by SEQ ID NOS:1-4, each sequence representing the genome's 5′or 3′ cyclization sequence in one of two broad classes of flavivirus,and (v) capable of forming with the flavivirus ssRNA genome, aheteroduplex structure characterized by a T_(m) of dissociation of atleast 45° C., and having disrupted base pairing between the virus' 5′and 3′ cyclization sequences, as evidenced by inhibition of viralreplication in the cells. The analog has various embodiments, includingthose described above with respect to the use of the analog in antiviraltherapy.

These and other objects and features of the invention will become morefully apparent when the following detailed description of the inventionis read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows 5′-end, 3′-end portion of a flavivirus genome, withsecondary structure shown and the 5′ and 3′ cyclization sequencesindicated by a box at the top of the figure; and

FIGS. 2A-2G show the backbone structures of various oligonucleotideanalogs with uncharged backbones;

FIGS. 3A-3D show the repeating subunit segment of exemplary morpholinooligonucleotides, designated 3A-3D;

FIGS. 4A and 4B are plots of the response of TBEV and WNV to increasingconcentrations of TBEV antisense (FIG. 4A) and scrambled-sequenceantisense (FIG. 4B); and

FIGS. 5A-5D are plots of the response of four Dengue virus serotypes toDEN antisense (SEQ ID NO:27).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The terms below, as used herein, have the following meanings, unlessindicated otherwise:

The term “oligonucleotide analog” refers to oligonucleotide having

(i) a modified backbone structure, e.g., a backbone other than thestandard phosphodiester linkage found in natural oligo- andpolynucleotides, and(ii) optionally, modified sugar moieties, e.g., morpholino moietiesrather than ribose or deoxyribose moieties. The analog supports basescapable of hydrogen bonding by Watson-Crick base pairing to standardpolynucleotide bases, where the analog backbone presents the bases in amanner to permit such hydrogen bonding in a sequence-specific fashionbetween the oligonucleotide analog molecule and bases in a standardpolynucleotide (e.g., single-stranded RNA or single-stranded DNA).Preferred analogs are those having a substantially uncharged, phosphoruscontaining backbone.

A substantially uncharged, phosphorus containing backbone in anoligonucleotide analog is one in which a majority of the subunitlinkages, e.g., between 60-100%, are uncharged at physiological pH, andcontain a single phosphorous atom. The analog contains between 8 and 40subunits, typically about 8-25 subunits, and preferably about 12 to 25subunits. The analog may have exact sequence complementarity to thetarget sequence or near complementarity, as defined below.

A “subunit” of an oligonucleotide analog refers to one nucleotide (ornucleotide analog) unit of the analog. The term may refer to thenucleotide unit with or without the attached intersubunit linkage,although, when referring to a “charged subunit”, the charge typicallyresides within the intersubunit linkage (e.g. a phosphate orphosphorothioate linkage).

A “morpholino oligonucleotide analog” is an oligonucleotide analogcomposed of morpholino subunit structures of the form shown in FIGS.3A-3D, where (i) the structures are linked together byphosphorus-containing linkages, one to three atoms long, joining themorpholino nitrogen of one subunit to the 5′ exocyclic carbon of anadjacent subunit, and (ii) P_(i) and P_(j) are purine or pyrimidinebase-pairing moieties effective to bind, by base-specific hydrogenbonding, to a base in a polynucleotide. The purine or pyrimidinebase-pairing moiety is typically adenine, cytosine, guanine, uracil orthymine. The synthesis, structures, and binding characteristics ofmorpholino oligomers are detailed in U.S. Pat. Nos. 5,698,685,5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and 5,506,337,all of which are incorporated herein by reference.

The subunit and linkage shown in FIG. 3B are used for six-atomrepeating-unit backbones, as shown in FIG. 3B (where the six atomsinclude: a morpholino nitrogen, the connected phosphorus atom, the atom(usually oxygen) linking the phosphorus atom to the 5′ exocyclic carbon,the 5′ exocyclic carbon, and two carbon atoms of the next morpholinoring). In these structures, the atom Y₁ linking the 5′ exocyclicmorpholino carbon to the phosphorus group may be sulfur, nitrogen,carbon or, preferably, oxygen. The X moiety pendant from the phosphorusis any stable group which does not interfere with base-specific hydrogenbonding. Preferred X groups include fluoro, alkyl, alkoxy, thioalkoxy,and alkyl amino, including cyclic amines, all of which can be variouslysubstituted, as long as base-specific bonding is not disrupted. Alkyl,alkoxy and thioalkoxy preferably include 1-6 carbon atoms. Alkyl aminopreferably refers to lower alkyl (C₁ to C₆) substitution, and cyclicamines are preferably 5- to 7-membered nitrogen heterocycles optionallycontaining 1-2 additional heteroatoms selected from oxygen, nitrogen,and sulfur. Z is sulfur or oxygen, and is preferably oxygen.

A preferred morpholino oligomer is a phosphorodiamidate-linkedmorpholino oligomer, referred to herein as a PMO. Such oligomers arecomposed of morpholino subunit structures such as shown in FIG. 3B,where X=NH₂, NHR, or NR₂ (where R is lower alkyl, preferably methyl),Y=O, and Z=O, and P_(i) and P_(j) are purine or pyrimidine base-pairingmoieties effective to bind, by base-specific hydrogen bonding, to a basein a polynucleotide. Also preferred are structures having an alternatephosphorodiamidate linkage, where, in FIG. 3B, X=lower alkoxy, such asmethoxy or ethoxy, Y=NH or NR, where R is lower alkyl, and Z=O.

The term “substituted”, particularly with respect to an alkyl, alkoxy,thioalkoxy, or alkylamino group, refers to replacement of a hydrogenatom on carbon with a heteroatom-containing substituent, such as, forexample, halogen, hydroxy, alkoxy, thiol, alkylthio, amino, alkylamino,imino, oxo (keto), nitro, cyano, or various acids or esters such ascarboxylic, sulfonic, or phosphonic. It may also refer to replacement ofa hydrogen atom on a heteroatom (such as an amine hydrogen) with analkyl, carbonyl or other carbon containing group.

As used herein, the term “target”, relative to the viral genomic RNA,refers to a viral genomic RNA, and may include either the positivestrand RNA which is the replicative strand of the virus, or the negativeor antisense strand which is formed in producing multiple new copies ofthe positive-strand RNA.

The term “target sequence” refers to a portion of the target RNA againstwhich the oligonucleotide analog is directed, that is, the sequence towhich the oligonucleotide analog will hybridize. The target sequenceincludes at least a portion of one of the sequences identified as SEQ IDNOS:1-4, representing the genome's 5′ or 3′ cyclization sequences in oneof two broad classes of flavivirus, as discussed further below. As willbe seen, the target sequence may be a contiguous region of the viralgenome, or may be composed of complementary fragments of both the 5′ and3′ cyclization sequences of the genome.

The term “targeting sequence” is the sequence in the oligonucleotideanalog that is complementary or substantially complementary to thetarget sequence in the RNA genome. The entire sequence, or only aportion of, the analog may be complementary to the target sequence, oronly a portion of the total analog sequence. For example, in an analoghaving 20 bases, only 8-12 may be targeting sequences. Typically, thetargeting sequence is formed of contiguous bases in the analog, but mayalternatively be formed of non-contiguous sequences that when placedtogether, e.g., from opposite ends of the analog, constitute a sequencethat spans the target sequence. As will be seen, the target andtargeting sequences are selected such that binding of the analog to theviral genome acts to disrupt or prevent formation of RNA secondarystructure formed by 5′- and 3′-cyclization sequences in the genome.

Target and targeting sequences are described as “complementary” to oneanother when hybridization occurs in an antiparallel configuration. Adouble-stranded polynucleotide can be “complementary” to anotherpolynucleotide. A targeting may have “near” or “substantial”complementarity to the target sequence and still function for thepurpose of the present invention. Preferably, the oligonucleotideanalogs employed in the present invention have at most one mismatch withthe target sequence out of 10 nucleotides, and preferably at most onemismatch out of 20. Alternatively, the antisense oligomers employed haveat least 90% sequence homology, and preferably at least 95% sequencehomology, with the exemplary targeting sequences as designated herein.

An oligonucleotide analog “specifically hybridizes” to a targetpolynucleotide if the oligomer hybridizes to the target underphysiological conditions, with a T_(m) substantially greater than 45°C., preferably at least 50° C., and typically 60° C.-80° C. or higher.Such hybridization preferably corresponds to stringent hybridizationconditions. At a given ionic strength and pH, the T_(m) is thetemperature at which 50% of a target sequence hybridizes to acomplementary polynucleotide. Again, such hybridization may occur with“near” or “substantial” complementarity of the antisense oligomer to thetarget sequence, as well as with exact complementarity.

A “nuclease-resistant” oligomeric molecule (oligomer) refers to onewhose backbone is substantially resistant to nuclease cleavage, innon-hybridized or hybridized form; by common extracellular andintracellular nucleases in the body; that is, the oligomer shows littleor no nuclease cleavage under normal nuclease conditions in the body towhich the oligomer is exposed.

A “heteroduplex refers to a duplex between an oligonucleotide analog andthe complementary portion of a target RNA. A “nuclease-resistantheteroduplex” refers to a heteroduplex formed by the binding of anantisense oligomer to its complementary target, such that theheteroduplex is substantially resistant to in vivo degradation byintracellular and extracellular nucleases, such as RNAseH, which arecapable of cutting double-stranded RNA/RNA or RNA/DNA complexes.

A “base-specific intracellular binding event involving a target RNA”refers to the specific binding of an oligonucleotide analog to a targetRNA sequence inside a cell. The base specificity of such binding issequence specific. For example, a single-stranded polynucleotide canspecifically bind to a single-stranded polynucleotide that iscomplementary in sequence.

An “effective amount” of an antisense oligomer, targeted against aninfecting ssRNA virus, is an amount effective to reduce the rate ofreplication of the infecting virus, and/or viral load, and/or symptomsassociated with the viral infection.

As used herein, the term “body fluid” encompasses a variety of sampletypes obtained from a subject including, urine, saliva, plasma, blood,spinal fluid, or other sample of biological origin, such as skin cellsor dermal debris, and may refer to cells or cell fragments suspendedtherein, or the liquid medium and its solutes.

The term “relative amount” is used where a comparison is made between atest measurement and a control measurement. The relative amount of areagent forming a complex in a reaction is the amount reacting with atest specimen, compared with the amount reacting with a controlspecimen. The control specimen may be run separately in the same assay,or it may be part of the same sample (for example, normal tissuesurrounding a malignant area in a tissue section).

“Treatment” of an individual or a cell is any type of interventionprovided as a means to alter the natural course of the individual orcell. Treatment includes, but is not limited to, administration of e.g.,a pharmaceutical composition, and may be performed eitherprophylactically, or subsequent to the initiation of a pathologic eventor contact with an etiologic agent. The related term “improvedtherapeutic outcome” relative to a patient diagnosed as infected with aparticular virus, refers to a slowing or diminution in the growth ofvirus, or viral load, or detectable symptoms associated with infectionby that particular virus.

An agent is “actively taken up by mammalian cells” when the agent canenter the cell by a mechanism other than passive diffusion across thecell membrane. The agent may be transported, for example, by “activetransport”, referring to transport of agents across a mammalian cellmembrane by e.g. an ATP-dependent transport mechanism, or by“facilitated transport”, referring to transport of antisense agentsacross the cell membrane by a transport mechanism that requires bindingof the agent to a transport protein, which then facilitates passage ofthe bound agent across the membrane. For both active and facilitatedtransport, the oligonucleotide analog preferably has a substantiallyuncharged backbone, as defined below. Alternatively, the antisensecompound may be formulated in a complexed form, such as an agent havingan anionic backbone complexed with cationic lipids or liposomes, whichcan be taken into cells by an endocytotic mechanism. The analog may beconjugated, e.g., at its 5′ or 3′ end, to an arginine rich peptide,e.g., the HIV TAT protein, or polyarginine, to facilitate transport intothe target host cell.

II. Target Flaviviruses

The present invention is based on the discovery that effectiveinhibition of flavivirus replication can be achieved by exposingflavivirus-infected cells to oligomeric analogs (i) targeted to the3′-CS (or 5′-CS) region of flaviviral RNA and (ii) having physical andpharmacokinetic features which allow effective interaction between theanalog and the viral RNA within host cells. In one aspect, the analogscan be used in treating a mammalian subject infected with the virus.

The invention targets members of the flavivirus genus of the flaviridaefamily including the viruses described below. Various physical,morphological, and biological characteristics of the flavivirus genes,and members therein, can be found, for example, in Textbook of HumanVirology, R. Belshe, ed., 2^(nd) Edition, Mosby, 1991, and in one ormore of the references cited above. Some of the key biological,pathological and epidemiological characteristics of each flavivirusmember are summarized below.

Flavivirus Replication

Flaviviruses are small, enveloped viruses containing a single,positive-strand, genomic RNA, approximately 10,500 nucleotides in lengthcontaining short 5′ and 3′ non-translated regions (NTRs), a single longopen reading frame, a 5′ cap, and a nonpolyadenylated 3′ terminus. Thecomplete nucleotide sequence of numerous flaviviral genomes, includingall four dengue serotypes, yellow fever virus, Japanese encephalitisvirus, West Nile virus and tick-borne encephalitis virus have beenreported. All flaviviral proteins are derived from a single longpolyprotein through precise processing events mediated by host as wellas virally encoded proteases. The ten gene products encoded by thesingle open reading frame are translated as a polyprotein organized inthe order, capsid (C), preMembrane (prM, which is processed to Membrane(M) just prior to virion release from the cell), Envelope (E) and theseven non-structural (NS) proteins: NS1, NS2a, NS2b, NS3, NS4a, NS4b andNS5 (Leyssen, De Clercq et al. 2000; Brinton 2002).

All the mosquito-borne flaviviruses share conserved RNA sequences andstructures (Proutski, Gould et al. 1997; Zeng, Falgout et al. 1998; Li,Li et al. 2002). Sequence comparison and RNA secondary structurepredictions of flavivirus 3′-NTR and 5′-NTR have revealed several short,well-conserved sequences and indicated that the 3′-terminal region(approximately 90 bases) can be folded in a conserved stem-loopstructure (Hahn, Hahn et al. 1987). Conserved stem loop structures havebeen shown to be important for viral replication in many positive-strandRNA viruses. The flavivirus 3′ stem-loop structure primary sequence isnot well conserved among flaviviruses but secondary structure is wellconserved. A short conserved sequence (3′-CS, SEQ ID NO: 3) has beenidentified upstream (i.e. in the 5′ direction) of the conservedstem-loop structure. Complementarity between the 3′-CS and a conservedsequence at the 5′ end of the genome (5′-CS, SEQ ID NO: 1) has beenproposed to result in a long-range intramolecular RNA interaction orcyclization of the genomic RNA (Hahn, Hahn et al. 1987; You, Falgout etal. 2001; Corver, Lenches et al. 2003). Recent experiments suggest thatbase-pairing between these sequences is essential for RNA replication ofa Kunjin virus replicon (Khromykh, Meka et al. 2001). Acomputer-generated predicted secondary-structure between the 5′-CS and3′-CS of Dengue virus is shown in FIG. 1 (Khromykh, Meka et al. 2001),with the 5′-CS and 3′-CS shown within a box in the upper portion of thefigure, and identified herein as SEQ ID NOS: 1 and 3, respectively.Short complementary sequences have also been identified at the 5′ and 3′end regions of tick-borne encephalitis viral genomes (SEQ ID NOS: 2 and4, respectively) and are proposed to function similarly as potentialcyclization sequences (Khromykh, Meka et al. 2001).

Dengue Virus

Although flavivirus transmission and the pathology of infection arequite varied among the different viruses, dengue viruses serve as anillustrative example of the genus. Dengue viruses are arthropod-borneviruses (arboviruses) and transmitted to man by mosquitoes of the genusAedes, primarily A. aegypti and A. albopictus. The viruses cause anillness manifested by high fever, headache, aching muscles and joints,and rash. In some cases, typically in children, a more severe form ofinfection is seen with dengue hemorrhagic fever/dengue shock syndrome(DHF/DSS), marked by severe hemorrhage, vascular permeability, or both,leading to shock. Individuals who have been infected with a given dengueserotype and are subsequently infected with a different serotype are atsignificantly greater risk for DHF/DSS. Without diagnosis and promptmedical intervention, the sudden onset and rapid progression of DHF/DSScan be fatal.

Endemic dengue caused by one or more of the four types of dengue virusesis a major public health problem in many tropical and subtropical areas.Sporadic dengue epidemics at times involving over a million individualscontinue to occur. Dengue viruses are the most significant group ofarthropod-transmitted viruses in terms of global morbidity and mortalitywith an estimated one hundred million cases of dengue fever occurringannually including 500,000 cases of DHF/DSS. With the global increase inpopulation, urbanization of the population especially throughout thetropics, and the lack of sustained mosquito control measures, themosquito vectors of dengue have expanded their distribution throughoutthe tropics, subtropics, and some temperate areas, bringing the risk ofdengue infection to over half the world's population. Modern jet traveland human emigration have facilitated global distribution of dengueserotypes such that multiple serotypes of dengue are now endemic in manyregions. Accompanying this there has been an increase in the frequencyof dengue epidemics and the incidence of DHF/DSS in the last 15 years.For example, in Southeast Asia, DHF/DSS is a leading cause ofhospitalization and death among children (Hayes and Gubler 1992).

As described below, many other members of the flavivirus genus are alsoetiologic agents of severe diseases such as yellow fever, Japaneseencephalitis, St. Louis encephalitis, Australian encephalitis, andtick-borne encephalitis.

Yellow Fever Virus

Although an effective vaccine for Yellow Fever has been available formany years, this virus continues to be a leading cause of hemorrhagicfever with mortality rates as high as 50%. Worldwide, there are 200,000estimated cases of yellow fever (with 30,000 deaths) annually. Smallnumbers of imported cases also occur in countries free of yellow fever(WHO, Fact Sheet 100, 2001).

Japanese Encephalitis Virus

This arbovirus is the leading cause of viral encephalitis worldwide.Approximately 50,000 cases occur annually in Asia and result in high(30%) mortality or in permanent neurological sequelae (30%) of patientswho survive. Epidemic outbreaks caused by JEV continue to pose seriouspublic health problems in the densely populated regions of tropical andsubtropical Asia. Transmitted by species of the Culex genus ofmosquitoes, the disease is clinically manifested as encephalitis, oftensevere and with a high mortality rate among young children and elderlypeople. JEV also infects domestic animals such as swine and horses.During the last two decades, immunization using an inactivated JEVvaccine has brought the disease under control in Japan, Korea andTaiwan. However, because of the high cost of manufacturing the vaccine,it is not readily available to those countries where it is needed themost (CDC, Japanese Encephalitis Fact Sheet, 2001).

Murray Valley Encephalitis Virus and Kunjin Virus

These viruses are the causative agents for “Australian encephalitis”, aclinical syndrome characterized by aseptic meningitis and/orencephalitis. Both are arboviruses, transmitted by the Culex mosquito,and are endemic in Northern Australia. Murray Valley encephalitissymptoms almost invariably include a sudden onset of fever; anorexia andheadache. Brain dysfunction may be experienced after a few days and bothcoma and death may ensue. It is rare for recovery from the encephaliticsyndrome to occur without some residual mental or functional disability.Kunjin virus, a virus closely related to West Nile virus (Scherret,Poidinger et al. 2001), causes a clinically similar illness to MurrayValley encephalitis virus but is generally less severe and humaninfections are reported less frequently.

West Nile Virus

West Nile virus (WNV), another arthropod-borne flavivirus, has emergedin recent years as a deadly health threat to not only humans, but alsoto other animal species such as horses and birds. In 1999, New York wasthe first area in North American to report cases of West Nile virusinfections. West Nile virus infection in humans has been foundpreviously only in Africa, the Middle East and Eastern Europe. The virusis transmitted to humans and several animal species by mosquitoes whichacquire the virus by feeding on infected birds. West Nile virus remainsa continued threat to public health. Epidemiologic and virologic studiesindicate that live virus persists in mosquito and bird populations.Mosquito control measures were implemented in New York, New Jersey andConnecticut, and many other Eastern states, yet new cases of West Nilevirus are being diagnosed. Currently, West Nile virus has been found innearly every state in the U.S. (Enserink 2002).

Among West Nile virus infected humans, approximately one in every 150 to300 become ill with fever, myalgia and possible rash. Among those whoare symptomatic, approximately 10-15% will have evidence of meningitis(headache, stiff neck) or encephalitis (change of mental status,peripheral neurologic abnormalities, muscle weakness). Almost allfatalities have occurred among humans over the age of 50. The fatalityrate among patients with central nervous system infection is between 5%and 11%. Fatalities have been due to prolonged central nervous systemdysfunction requiring ventilatory support and leading to secondarycomplications. Prolonged neurologic symptoms have occurred in survivorsof West Nile virus-caused encephalitis.

St. Louis Encephalitis Virus

Although there have been no recent epidemics of St. Louis Encephalitisvirus, it remains endemic in the western United States and is the causeof severe disease including asceptic meningitis and/or encephalitis.Another arbovirus, SLEV is responsible for unpredictable andintermittent epidemics with the largest recent U.S. outbreaks occurringin 1975 and 1990.

Tick-Borne Encephalitis Tick-borne encephalitis (TBE) is one of the mostdangerous human infections occurring in Europe and many parts of Asia.The etiological agent is Tick-borne encephalitis virus (TBEV). TBEV isbelieved to cause at least 11,000 human cases of encephalitis in Russia,including 89-166 deaths, and about 3000 cases in the rest of Europeannually (Ternovoi, Kurzhukov et al. 2003). TBE viruses are oftenvirulent with symptoms ranging from mild or asymptomatic to severeencephalitis with a mortality rate that approaches 40%. Related viruseswithin the same group, Louping ill virus (LIV) and Powassan virus (POW),also cause human encephalitis but rarely on an epidemic scale. Two otherviruses within the same group, Kyasanur Forest disease virus (KFD) andAlkhurma virus (ALK), are closely related to the TBE complex viruses andtend to cause fatal hemorrhagic fevers rather than encephalitis(Gritsun, Lashkevich et al. 2003).

III. Viral Target Regions and Targeting Sequences

The preferred target sequences are those adjacent and including at leasta portion, e.g., at least 2-8 bases, of the 5′-CS or 3′-CS sequences ofthe positive strand flaviviral RNA. As discussed above, these conservedcyclization sequences (CS) appear to play a role in viral replication bybringing into close proximity the 3′ and 5′ regions of the viral RNA inorder to initiate RNA replication (Hahn, Hahn et al. 1987; Khromykh,Meka et al. 2001). A variety of flaviviral genome sequences areavailable from well known sources, such as the NCBI GenBank databases.Alternatively, a person skilled in the art can find sequences for manyof the subject viruses in the open literature, e.g., by searching forreferences that disclose sequence information on designated viruses.Once a complete or partial viral sequence is obtained, the 5′ and 3′ CSsequences of the virus are identified.

GenBank references for exemplary viral nucleic acid sequences containingthe 5′-CS and 3′-CS end terminal sequences in the corresponding viralgenomes are listed in Table 1 below. It will be appreciated that thesesequences are only illustrative of other sequences in the flavirusgenus, as may be available from available gene-sequence databases ofliterature or patent resources. The sequences below, identified as SEQID NOS: 5-15, are also listed in Table 3 at the end of thespecification. The conserved 5′-CS and 3′-CS are shown in bold in Table1 and listed as SEQ ID NOS: 1-4 in Table 1.

Another group of exemplary target sequences are the complements of SEQID NOS: 5-15; that is the sequences that have the complementaryanti-parallel sequence of one of identified sequences 5-15. As anexample, the complement of SEQ ID NO: 5 (5′-GUCAAUAUGCUAAAACGCGG-3′) is5′-CCGCGUUUUAGCAUAUUGAC-3.

TABLE 1 Exemplary 5′-CS and 3′-CS Target Sequences GenBank 5′-CS SEQ IDTarget Sequence (5′ Virus Acc. No. Target Ncts. NO. to 3′) St. Louisencephalitis M16614 138-157 5 GUCAAUAUGCUA Murray Valley AF161266135-154 AAACGCGG encephalitis West Nile M12294 136-155 Kunjin D00246115-134 Japanese encephalitis M18370 135-154 6 AUCAAUAUGCUG AAACGCGGYellow fever X03700 155-174 7 GUCAAUAUGGUA CGACGAGG Dengue-Type 1 M87512115-135 8 CUUUCAAUAUGC Dengue-Type 2 M19197 131-151 UGAAACGCGDengue-Type 4 M14931 133-153 Dengue-Type 3 M93130 129-149 9 CUAUCAAUAUGCUGAAACGCG Tick borne encephalitis U27495 108-129 10 CAGCUUAGGAGAPowassen L06436  81-102 ACAAGAGCUG Louping III Y07863 105-126 KyasanurForest disease X74111  94-115 Alkhurma AF331718  90-111 West Nile M12294132-151 11 GGCUGUCAAUAU GCUAAAAC St. Louis encephalitis M1837010861-10882 12 AACAGCAUAUUG Japanese encephalitis ACACCUGGGA MurrayValley AF161266 10899-10920 encephalitis West Nile M12294 10853-10874Kunjin AY274505 10914-10934 Yellow fever X03700 10745-10767 13UGGGACCAUAUU GACGCCAGGGA Dengue-Type 1 M87512 10609-10630 14AAACAGCAUAUU Dengue-Type 2 M19197 10595-10616 GACGCUGGGA Dengue-Type 3M93130 10588-10609 Dengue-Type 4 M14931 10540-10561 Tick borneencephalitis U27495 11057-11078 15 CGGUUCUUGUUC Powassen L0643610755-10776 UCCCUGAGCC Louping III Y07863 10787-10808

The degree of complementarity between the target and targeting sequenceis sufficient to form a stable duplex. The region of complementarity ofthe antisense oligomers with the target RNA sequence may be as short as8-11 bases, but is preferably 12-15 bases or more, e.g. 12-20 bases, or12-25 bases. An antisense oligomer of about 15 bases is generally longenough to have a unique complementary sequence in the viral genome. Inaddition, a minimum length of complementary bases may be required toachieve the requisite binding T_(m), as discussed below.

Oligomers as long as 40 bases may be suitable, where at least theminimum number of bases, e.g., 8-11, preferably 12-15 bases, arecomplementary to the target sequence. In general, however, facilitatedor active uptake in cells is optimized at oligomer lengths less thanabout 30, preferably less than 25, and more preferably 20 or fewerbases. For PMO oligomers, described further below, an optimum balance ofbinding stability and uptake generally occurs at lengths of 13-23 bases.

The oligomer may be 100% complementary to the viral nucleic acid targetsequence, or it may include mismatches, e.g., to accommodate variants,as long as a heteroduplex formed between the oligomer and viral nucleicacid target sequence is sufficiently stable to withstand the action ofcellular nucleases and other modes of degradation which may occur invivo. Oligomer backbones which are less susceptible to cleavage bynucleases are discussed below. Mismatches, if present, are lessdestabilizing toward the end regions of the hybrid duplex than in themiddle. The number of mismatches allowed will depend on the length ofthe oligomer, the percentage of G:C base pairs in the duplex, and theposition of the mismatch(es) in the duplex, according to well understoodprinciples of duplex stability. Although such an antisense oligomer isnot necessarily 100% complementary to the viral nucleic acid targetsequence, it is effective to stably and specifically bind to the targetsequence, such that a biological activity of the nucleic acid target,e.g. cyclization of the viral RNA, is modulated.

The stability of the duplex formed between the oligomer and the targetsequence is a function of the binding T_(m) and the susceptibility ofthe duplex to cellular enzymatic cleavage. The T_(m) of an antisensecompound with respect to complementary-sequence RNA may be measured byconventional methods, such as those described by Hames et al., NucleicAcid Hybridization, IRL Press, 1985, pp. 107-108. Each antisenseoligomer should have a binding T_(m), with respect to acomplementary-sequence RNA, of greater than body temperature andpreferably greater than 45° C. T_(m)'s in the range 60-80° C. or greaterare preferred. According to well known principles, the T_(m) of anoligomer compound, with respect to a complementary-based RNA hybrid, canbe increased by increasing the ratio of C:G paired bases in the duplex,and/or by increasing the length (in base pairs) of the heteroduplex. Atthe same time, for purposes of optimizing cellular uptake, it may beadvantageous to limit the size of the oligomer. For this reason,compounds that show high T_(m) (50° C. or greater) at a length of 15bases or less are generally preferred over those requiring 20+ bases forhigh T_(m) values.

Table 2 below lists exemplary targeting sequences directed against the5′-CS and 3′-CS for selected viruses of the flavivirus genus. Thesesequences, identified by SEQ ID NOS:16-27, are complementary andantiparallel to the sequences identified as SEQ ID NOS:5-15 above (SEQID NO:19 is complementary to SEQ ID NO:8, and complementary with onemismatch to SEQ ID NO:9, SEQ ID NO:27 is targeted to a Dengue Type 2virus 3′-cyclization sequence). As noted above, the actual targetsequence in the oligonucleotide analog may be complementary to only aportion of the corresponding target sequence in Table 1, including aportion of the sequence in SEQ ID NO:1 or its complement SEQ ID NO:3, orSEQ ID NO:2 or its complement SEQ ID NO:4.

More generally, the invention contemplates, as exemplary targetingsequences, a sequence of at least 8 bases complementary to a region ofthe virus' positive strand RNA genome that includes at least a portionof the genome's 5′ or 3′ cyclization sequence SEQ ID NO:1 or itscomplement SEQ ID NO:3 for the group of flavivirus identified with this5′-CS, or SEQ ID NO:2 or its complement SEQ ID NO:4 for the group offlavivirus identified with this 5′-CS. In a preferred embodiment, thetargeting sequence is complementary to at least a portion of thegenome's 3′ cyclization sequence identified as SEQ ID NO:3 for one groupof flaviviruses, and as SEQ ID NO: 4 for another group of flaviviruses.The targeting sequence contains a sufficient number of bases in the CSsequence to disrupt base pairing between the virus' 5′ and 3′cyclization sequences, that is disrupt the paired-base stem secondarystructure shown in the cyclization box as illustrated in FIG. 1. Thenumber of targeting sequences needed to disrupt this structure ispreferably at least 2-4 bases complementary to one of the twocomplementary cyclization sequences, plus bases complementary toadjacent target-sequence bases.

In one embodiment, the targeting sequence includes bases complementaryto the entire 5′ or 3′ cyclization sequence of the selected virus, i.e.,any of SEQ ID NOS:1-4.

In another embodiment, the targeting sequence is complementary tocorresponding complementary regions of the two cyclization sequences. Asan example, an 8-base target sequence containing complementary 4-baseportions of the two cyclization sequences shown at the top of thesequence box in FIG. 1 has the discontinuous sequence 5′CAUA . . .UAUG3′. A targeting sequence effective to bind to and disrupt thissequence would have the sequence 5′CATA . . . TATG3′, where “ . . . ”could be a direct 5′-3′ subunit link, or a spacer, as a PEG linker,designed to accommodate the discontinuity in the target sequence.

The latter embodiment represents a specific case of an antisenseoligonucleotide analog, in accordance with another aspect of theinvention, that is directed to complementary portions of sequencesforming a “stem” secondary structure in RNA, and includes as targetingbases, bases complementary to both strands of the stem structure, forpurposes of disrupting the stem structure.

TABLE 2 Exemplary Targeting Sequences Against the 5′-CS and 3′-CS ofFlaviviruses SEQ GenBank 5′-CS Targeting Sequences ID Virus Acc. No.(5′ to 3′) NO. St. Louis encephalitis M16614 CCGCGTTTTAGCATATTGAC 16Murray Valley encephalitis AF161266 West Nile M12294 Kunjin D00246Japanese encephalitis M18370 CCGCGTTTCAGCATATTGAT 17 Yellow fever X03700CCTCGTCGTACCATATTGAC 18 Dengue-Type 1 M87512 CGCGTTTCAGCATATTGAAAG 19Dengue-Type 2 M19197 Dengue-Type 3 M93130 Dengue-Type 4 M14931 Tickborne encephalitis U27495 CAGCTCTTGTTCTCCTAAGCTG 20 Powassen L06436Louping III Y07863 Kyasanu Forest disease X74111 Alkhurma AF331718 WestNile M12294 GTTTTAGCATATTGACAGCC 21 SEQ GenBank 3′-CS TargetingSequences ID Virus Acc. No. (5′ to 3′) NO. St. Louis encephalitisTCCCAGGTGTCAATATGCTGTT 22 Japanese encephalitis M18370 Murray Valleyencephalitis AF161266 West Nile M12294 Kunjin AY274505 Yellow feverX03700 TCCCTGGCGTCAATATGGTCCCA 23 Dengue-Type 1 M87512TCCCAGCGTCAATATGCTGTTT 24 Dengue-Type 2 M19197 Dengue-Type 3 M93130Dengue-Type 4 M14931 Tick borne encephalitis U27495GGCTCAGGGAGAACAAGAACCG 25 Powassen L06436 Louping III Y07863 West NileM12294 CAGGTGTCAATATGCTGTTTTG 26 Dengue-Type 2 M19197 CCCAGCGTCAATATGCTG27

Note that the target sequence in Table 1 is indicated as containinguracil (U) bases characteristic of RNA, and the targeting sequences inTable 2, as containing thymine bases characteristic of DNA. It will beunderstood that the targeting sequence bases may be normal DNA bases oranalogs thereof, e.g., uracil, that are capable of Watson-Crick basepairing to target-sequence RNA bases.

IV. Antisense Oligomers

A. Properties

As detailed above, the antisense oligomer has a base sequence directedto a targeted portion of the viral genome, preferably either the 5′-CSor 3′-CS. In addition, the oligomer is able to effectively targetinfecting viruses, when administered to an infected host cell, e.g. inan infected animal subject. This requirement is met when the oligomercompound (a) has the ability to be actively taken up by mammalian cells,and (b) once taken up, form a duplex with the target ssRNA with a T_(m)greater than about 50° C.

As will be described below, the ability to be taken up by cells requiresthat the oligomer backbone be substantially uncharged, and, preferably,that the oligomer structure is recognized as a substrate for active orfacilitated transport across the cell membrane. The ability of theoligomer to form a stable duplex with the target RNA will also depend onthe oligomer backbone, as well as factors noted above, the length anddegree of complementarity of the antisense oligomer with respect to thetarget, the ratio of G:C to A:T base matches, and the positions of anymismatched bases. The ability of the antisense oligomer to resistcellular nucleases promotes survival and ultimate delivery of the agentto the cell cytoplasm.

Below are disclosed methods for testing any given, substantiallyuncharged backbone for its ability to meet these requirements.

A1. Active or Facilitated Uptake by Cells

The antisense compound may be taken up by host cells by facilitated oractive transport across the host cell membrane if administered in free(non-complexed) form, or by an endocytotic mechanism if administered incomplexed form.

In the case where the agent is administered in free form, the antisensecompound should be substantially uncharged, meaning that a majority ofits intersubunit linkages are uncharged at physiological pH. Experimentscarried out in support of the invention indicate that a small number ofnet charges, e.g., 1-2 for a 15- to 20-mer oligomer, can in fact enhancecellular uptake of certain oligomers with substantially unchargedbackbones. The charges may be carried on the oligomer itself, e.g., inthe backbone linkages, or may be terminal charged-group appendages.Preferably, the number of charged linkages is no more than one chargedlinkage per four uncharged linkages. More preferably, the number is nomore than one charged linkage per ten, or no more than one per twenty,uncharged linkages. In one embodiment, the oligomer is fully uncharged.

An oligomer may also contain both negatively and positively chargedbackbone linkages, as long as opposing charges are present inapproximately equal number. Preferably, the oligomer does not includeruns of more than 3-5 consecutive subunits of either charge. Forexample, the oligomer may have a given number of anionic linkages, e.g.phosphorothioate or N3′→P5′ phosphoramidate linkages, and a comparablenumber of cationic linkages, such as N,N-diethylenediaminephosphoramidates (Dagle, Littig et al. 2000). The net charge ispreferably neutral or at most 1-2 net charges per oligomer.

In addition to being substantially or fully uncharged, the antisenseagent is preferably a substrate for a membrane transporter system (i.e.,a membrane protein or proteins) capable of facilitating transport oractively transporting the oligomer across the cell membrane. Thisfeature may be determined by one of a number of tests for oligomerinteraction or cell uptake, as follows.

A first test assesses binding at cell surface receptors, by examiningthe ability of an oligomer compound to displace or be displaced by aselected charged oligomer, e.g., a phosphorothioate oligomer, on a cellsurface. The cells are incubated with a given quantity of test oligomer,which is typically fluorescently labeled, at a final oligomerconcentration of between about 10-300 nM. Shortly thereafter, e.g.,10-30 minutes (before significant internalization of the test oligomercan occur), the displacing compound is added, in incrementallyincreasing concentrations. If the test compound is able to bind to acell surface receptor, the displacing compound will be observed todisplace the test compound. If the displacing compound is shown toproduce 50% displacement at a concentration of 10× the test compoundconcentration or less, the test compound is considered to bind at thesame recognition site for the cell transport system as the displacingcompound.

A second test measures cell transport, by examining the ability of thetest compound to transport a labeled reporter, e.g., a fluorescencereporter, into cells. The cells are incubated in the presence of labeledtest compound, added at a final concentration between about 10-300 nM.After incubation for 30-120 minutes, the cells are examined, e.g., bymicroscopy, for intracellular label. The presence of significantintracellular label is evidence that the test compound is transported byfacilitated or active transport.

The antisense compound may also be administered in complexed form, wherethe complexing agent is typically a polymer, e.g., a cationic lipid,polypeptide, or non-biological cationic polymer, having an oppositecharge to any net charge on the antisense compound. Methods of formingcomplexes, including bilayer complexes, between anionic oligonucleotidesand cationic lipid or other polymer components, are well known. Forexample, the liposomal composition Lipofectin® (Feigner, Gadek et al.1987), containing the cationic lipid DOTMA(N-[1-(2,3-dioleyloxy)propyI]-N,N,N-trimethylammonium chloride) and theneutral phospholipid DOPE (dioleyl phosphatidyl ethanolamine), is widelyused. After administration, the complex is taken up by cells through anendocytotic mechanism, typically involving particle encapsulation inendosomal bodies.

The antisense compound may also be administered in conjugated form withan arginine-rich peptide linked to the 5′ or 3′ end of the antisenseoligomer. The peptide is typically 8-16 amino acids and consists of amixture of arginine, and other amino acids including phenyalanine andcysteine. Exposure of cells to the peptide conjugated oligomer resultsin enhanced intracellular uptake and delivery to the RNA target(Moulton, Nelson et al. 2004).

Alternatively, and according to another aspect of the invention, therequisite properties of oligomers with any given backbone can beconfirmed by a simple in vivo test, in which a labeled compound isadministered to an animal, and a body fluid sample, taken from theanimal several hours after the oligomer is administered, assayed for thepresence of heteroduplex with target RNA. This method is detailed insubsection D below.

A2. Substantial Resistance to RNaseH

Two general mechanisms have been proposed to account for inhibition ofexpression by antisense oligonucleotides (Agrawal, Mayrand et al. 1990;Bonham, Brown et al. 1995; Boudvillain, Guerin et al. 1997). In thefirst, a heteroduplex formed between the oligonucleotide and the viralRNA acts as a substrate for RNaseH, leading to cleavage of the viralRNA. Oligonucleotides belonging, or proposed to belong, to this classinclude phosphorothioates, phosphotriesters, and phosphodiesters(unmodified “natural” oligonucleotides). Such compounds expose the viralRNA in an oligomer:RNA duplex structure to hydrolysis by RNaseH, andtherefore loss of function.

A second class of oligonucleotide analogs, termed “steric blockers” or,alternatively, “RNaseH inactive” or “RNaseH resistant”, have not beenobserved to act as a substrate for RNaseH, and are believed to act bysterically blocking target RNA nucleocytoplasmic transport, splicing,translation, or replication. This class includes methylphosphonates(Toulme, Tinevez et al. 1996), morpholino oligonucleotides, peptidenucleic acids (PNA's), certain 2′-O-allyl or 2′-O-alkyl modifiedoligonucleotides (Bonham, Brown et al. 1995), and N3′→P5′phosphoramidates (Ding, Grayaznov et al. 1996; Gee, Robbins et al.1998).

A test oligomer can be assayed for its RNaseH resistance by forming anRNA:oligomer duplex with the test compound, then incubating the duplexwith RNaseH under standard assay conditions, as described in Stein etal. After exposure to RNaseH, the presence or absence of intact duplexcan be monitored by gel electrophoresis or mass spectrometry.

A3. In Vivo Uptake

In accordance with another aspect of the invention, there is provided asimple, rapid test for confirming that a given antisense oligomer typeprovides the required characteristics noted above, namely, high T_(m),ability to be actively taken up by the host cells, and substantialresistance to RNaseH. This method is based on the discovery that aproperly designed antisense compound will form a stable heteroduplexwith the complementary portion of the viral RNA target when administeredto a mammalian subject, and the heteroduplex subsequently appears in theurine (or other body fluid). Details of this method are also given inco-owned U.S. patent application Ser. No. 09/736,920, entitled“Non-Invasive Method for Detecting Target RNA” (Non-Invasive Method),the disclosure of which is incorporated herein by reference.

Briefly, a test oligomer containing a backbone to be evaluated, having abase sequence targeted against a known RNA, is injected into an animal,e.g., mammalian subject. The antisense oligomer may be directed againstany intracellular RNA, including a host RNA or the RNA of an infectingvirus. Several hours (typically 8-72) after administration, the urine isassayed for the presence of the antisense-RNA heteroduplex. Ifheteroduplex is detected, the backbone is suitable for use in theantisense oligomers of the present invention.

The test oligomer may be labeled, e.g. by a fluorescent or a radioactivetag, to facilitate subsequent analyses, if it is appropriate for themammalian subject. The assay can be in any suitable solid-phase or fluidformat. Generally, a solid-phase assay involves first binding theheteroduplex analyte to a solid-phase support, e.g., particles or apolymer or test-strip substrate, and detecting the presence/amount ofheteroduplex bound. In a fluid-phase assay, the analyte sample istypically pretreated to remove interfering sample components. If theoligomer is labeled, the presence of the heteroduplex is confirmed bydetecting the label tags. For non-labeled compounds, the heteroduplexmay be detected by immunoassay if in solid phase format or by massspectroscopy or other known methods if in solution or suspension format.

When the antisense oligomer is complementary to a virus-specific regionof the viral genome (such as the region encompassing the flavivirus5′-CS and 3′-CS), the method can be used to detect the presence of agiven ssRNA virus. The method can also be used to monitor the reductionin the amount of virus during a treatment method.

B. Exemplary Oligomer Backbones

Examples of nonionic linkages that may be used in oligonucleotideanalogs are shown in FIGS. 2A-2G. In these figures, B represents apurine or pyrimidine base-pairing moiety effective to bind, bybase-specific hydrogen bonding, to a base in a polynucleotide,preferably selected from adenine, cytosine, guanine and uracil. Suitablebackbone structures include carbonate (2A, R=O) and carbamate (2A,R=NH₂) linkages (Mertes and Coats 1969; Gait, Jones et al. 1974); alkylphosphonate and phosphotriester linkages (2B, R=alkyl or —O-alkyl)(Lesnikowski, Jaworska et al. 1990); amide linkages (2C) (Blommers,Pieles et al. 1994); sulfone and sulfonamide linkages (2D, R₁, R₂=CH₂)(Roughten, 1995; McElroy, 1994); and a thioformacetyl linkage (2E)(Matteucci, 1990; Cross, 1997). The latter is reported to have enhancedduplex and triplex stability with respect to phosphorothioate antisensecompounds (Cross, 1997). Also reported are the3′-methylene-N-methylhydroxyamino compounds of structure 2F (Mohan,1995).

Peptide nucleic acids (PNAs) (FIG. 2G) are analogs of DNA in which thebackbone is structurally homomorphous with a deoxyribose backbone,consisting of N-(2-aminoethyl) glycine units to which pyrimidine orpurine bases are attached. PNAs containing natural pyrimidine and purinebases hybridize to complementary oligonucleotides obeying Watson-Crickbase-pairing rules, and mimic DNA in terms of base pair recognition(Egholm et al., 1993). The backbone of PNAs is formed by peptide bondsrather than phosphodiester bonds, making them well-suited for antisenseapplications. The backbone is uncharged, resulting in PNA/DNA or PNA/RNAduplexes which exhibit greater than normal thermal stability. PNAs arenot recognized by nucleases or proteases.

A preferred oligomer structure employs morpholino-based subunits bearingbase-pairing moieties, joined by uncharged linkages, as described above.Especially preferred is a substantially unchargedphosphorodiamidate-linked morpholino oligomer, such as illustrated inFIGS. 3A-3D. Morpholino oligonucleotides, including antisense oligomers,are detailed, for example, in co-owned U.S. Pat. Nos. 5,698,685,5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,185,444, 5,521,063, and5,506,337, all of which are expressly incorporated by reference herein.

Important properties of the morpholino-based subunits include: theability to be linked in an oligomeric form by stable, uncharged backbonelinkages; the ability to support a nucleotide base (e.g. adenine,cytosine, guanine or uracil) such that the polymer formed can hybridizewith a complementary-base target nucleic acid, including target RNA,with high T_(m), even with oligomers as short as 10-14 bases; theability of the oligomer to be actively transported into mammalian cells;and the ability of the oligomer:RNA heteroduplex to resist RNAsedegradation.

Exemplary backbone structures for antisense oligonucleotides of theinvention include the β-morpholino subunit types shown in FIGS. 3A-3D,each linked by an uncharged, phosphorus-containing subunit linkage. FIG.3A shows a phosphorus-containing linkage which forms the five atomrepeating-unit backbone, where the morpholino rings are linked by a1-atom phosphoamide linkage. FIG. 3B shows a linkage which produces a6-atom repeating-unit backbone. In this structure, the atom Y linkingthe 5′ morpholino carbon to the phosphorus group may be sulfur,nitrogen, carbon or, preferably, oxygen. The X moiety pendant from thephosphorus may be fluorine, an alkyl or substituted alkyl, an alkoxy orsubstituted alkoxy, a thioalkoxy or substituted thioalkoxy, orunsubstituted, monosubstituted, or disubstituted nitrogen, includingcyclic structures, such as morpholines or piperidines. Alkyl, alkoxy andthioalkoxy preferably include 1-6 carbon atoms. The Z moieties aresulfur or oxygen, and are preferably oxygen.

The linkages shown in FIGS. 3C and 3D are designed for 7-atomunit-length backbones. In Structure 3C, the X moiety is as in Structure3B, and the moiety Y may be methylene, sulfur, or, preferably, oxygen.In Structure 3D, the X and Y moieties are as in Structure 3B.Particularly preferred morpholino oligonucleotides include thosecomposed of morpholino subunit structures of the form shown in FIG. 3B,where X=NH₂ or N(CH₃)₂, Y=O, and Z=O.

As noted above, the substantially uncharged oligomer may advantageouslyinclude a limited number of charged linkages, e.g. up to about 1 perevery 5 uncharged linkages, more preferably up to about 1 per every 10uncharged linkages. Therefore a small number of charged linkages, e.g.charged phosphoramidate or phosphorothioate, may also be incorporatedinto the oligomers.

The antisense compounds can be prepared by stepwise solid-phasesynthesis, employing methods detailed in the references cited above. Insome cases, it may be desirable to add additional chemical moieties tothe antisense compound, e.g. to enhance pharmacokinetics or tofacilitate capture or detection of the compound. Such a moiety may becovalently attached, typically to a terminus of the oligomer, accordingto standard synthetic methods. For example, addition of apolyethyleneglycol moiety or other hydrophilic polymer, e.g., one having10-100 monomeric subunits, may be useful in enhancing solubility. One ormore charged groups, e.g., anionic charged groups such as an organicacid, may enhance cell uptake. A reporter moiety, such as fluorescein ora radiolabeled group, may be attached for purposes of detection.Alternatively, the reporter label attached to the oligomer may be aligand, such as an antigen or biotin, capable of binding a labeledantibody or streptavidin. In selecting a moiety for attachment ormodification of an antisense oligomer, it is generally of coursedesirable to select chemical compounds of groups that are biocompatibleand likely to be tolerated by a subject without undesirable sideeffects.

V. Inhibition of Viral Replication

The antisense compounds detailed above are useful in inhibitingreplication of flaviviruses in animal cells, including mammalian cells,e.g., human cells, and avian cells. In one embodiment, such inhibitionis effective in treating infection of a host animal by these viruses.Accordingly, the method comprises, in one embodiment, contacting a cellinfected with the virus with an antisense agent effective to inhibit thereplication of the specific virus. In this embodiment, the antisenseagent is administered to a mammalian subject, e.g., human or domesticanimal, infected with a given virus, in a suitable pharmaceuticalcarrier. It is contemplated that the antisense oligonucleotide arreststhe growth of the RNA virus in the host. The RNA virus may be decreasedin number or eliminated with little or no detrimental effect on thenormal growth or development of the host.

A. Identification of the Infective Agent

The specific virus causing the infection can be determined by methodsknown in the art, e.g. serological or cultural methods, or by methodsemploying the antisense oligomers of the present invention.

Serological identification employs a viral sample or culture isolatedfrom a biological specimen, e.g., stool, urine, cerebrospinal fluid,blood, etc., of the subject. Immunoassay for the detection of virus isgenerally carried out by methods routinely employed by those of skill inthe art, e.g., ELISA or Western blot. In addition, monoclonal antibodiesspecific to particular viral strains or species are often commerciallyavailable.

Culture methods may be used to isolate and identify particular types ofvirus, by employing techniques including, but not limited to, comparingcharacteristics such as rates of growth and morphology under variousculture conditions.

Another method for identifying the viral infective agent in an infectedsubject employs one or more antisense oligomers targeting a spectrum offlavivirus species. Sequences targeting any characteristic viral RNA canbe used. The desired target sequences are preferably (i) common to broadvirus families/genera, and (ii) not found in the infected host, e.g.,humans. Characteristic nucleic acid sequences for a large number ofinfectious viruses are available in public databases, and may serve asthe basis for the design of specific oligomers.

For each plurality of oligomers, the following steps are carried out:(a) the oligomer(s) are administered to the subject; (b) at a selectedtime after said administering, a body fluid sample is obtained from thesubject; and (c) the sample is assayed for the presence of anuclease-resistant heteroduplex comprising the antisense oligomer and acomplementary portion of the viral genome. Steps (a)-(c) are carried forat least one such oligomer, or as many as is necessary to identify thevirus or family of viruses. Oligomers can be administered and assayedsequentially or, more conveniently, concurrently. The virus isidentified based on the presence (or absence) of a heteroduplexcomprising the antisense oligomer and a complementary portion of theviral genome of the given known virus or family of viruses.

Preferably, a first group of oligomers, targeting broad families, isutilized first, followed by selected oligomers complementary to specificgenera and/or species and/or strains within the broad family/genusthereby identified. This second group of oligomers includes targetingsequences directed to specific genera and/or species and/or strainswithin a broad family/genus. Several different second oligomercollections, i.e. one for each broad virus family/genus tested in thefirst stage, are generally provided. Sequences are selected which are(i) specific for the individual genus/species/strains being tested and(ii) not found in humans.

B. Administration of the Antisense Oligomer

Effective delivery of the antisense oligomer to the target nucleic acidis an important aspect of treatment. In accordance with the invention,routes of antisense oligomer delivery include, but are not limited to,various systemic routes, including oral and parenteral routes, e.g.,intravenous, subcutaneous, intraperitoneal, and intramuscular, as wellas inhalation, transdermal and topical delivery. The appropriate routemay be determined by one of skill in the art, as appropriate to thecondition of the subject under treatment. For example, an appropriateroute for delivery of an antisense oligomer in the treatment of a viralinfection of the skin is topical delivery, while delivery of anantisense oligomer for the treatment of a viral respiratory infection isby inhalation. The oligomer may also be delivered directly to the siteof viral infection, or to the bloodstream.

The antisense oligomer may be administered in any convenient vehiclewhich is physiologically acceptable. Such a composition may include anyof a variety of standard pharmaceutically accepted carriers employed bythose of ordinary skill in the art. Examples include, but are notlimited to, saline, phosphate buffered saline (PBS), water, aqueousethanol, emulsions, such as oil/water emulsions or triglycerideemulsions, tablets and capsules. The choice of suitable physiologicallyacceptable carrier will vary dependent upon the chosen mode ofadministration.

In some instances, liposomes may be employed to facilitate uptake of theantisense oligonucleotide into cells. (See, e.g., Williams, S. A.,Leukemia 10(12)1980-1989, 1996; Lappalainen et al., Antiviral Res.23:119, 1994; Uhlmann et al., ANTISENSE OLIGONUCLEOTIDES: A NEWTHERAPEUTIC PRINCIPLE, Chemical Reviews, Volume 90, No. 4, pages544-584, 1990; Gregoriadis, G., Chapter 14, Liposomes, Drug Carriers inBiology and Medicine, pp. 287-341, Academic Press, 1979). Hydrogels mayalso be used as vehicles for antisense oligomer administration, forexample, as described in WO 93/01286. The oligonucleotides may also beadministered in microspheres or microparticles. (See, e.g., Wu, G. Y.and Wu, C. H., J. Biol. Chem. 262:4429-4432, 1987). Alternatively, theuse of gas-filled microbubbles complexed with the antisense oligomerscan enhance delivery to target tissues, as described in U.S. Pat. No.6,245,747.

Sustained release compositions may also be used. These may includesemipermeable polymeric matrices in the form of shaped articles such asfilms or microcapsules.

In one aspect of the method, the subject is a human subject, e.g., apatient diagnosed as having a localized or systemic viral infection. Thecondition of a patient may also dictate prophylactic administration ofan antisense oligomer of the invention, e.g. in the case of a patientwho (1) is immunocompromised; (2) is a burn victim; (3) has anindwelling catheter; or (4) is about to undergo or has recentlyundergone surgery. In one preferred embodiment, the oligomer is aphosphorodiamidate morpholino oligomer, contained in a pharmaceuticallyacceptable carrier, and is delivered orally. In another preferredembodiment, the oligomer is a phosphorodiamidate morpholino oligomer,contained in a pharmaceutically acceptable carrier, and is deliveredintravenously (IV).

In another application of the method, the subject is a livestock animal,e.g., a chicken, turkey, pig, cow or goat, etc., and the treatment iseither prophylactic or therapeutic. In other applications, the infectedanimal to be treated may be a zoo or wild animal, e.g., seal, penguin,or hawk, such to one or more flavivirus infections. The invention alsoincludes a livestock and poultry food composition containing a foodgrain supplemented with a subtherapeutic amount of an antiviralantisense compound of the type described above. Also contemplated is, ina method of feeding livestock and poultry with a food grain supplementedwith subtherapeutic levels of an antiviral, an improvement in which thefood grain is supplemented with a subtherapeutic amount of an antiviraloligonucleotide composition as described above.

The antisense compound is generally administered in an amount and mannereffective to result in a peak blood concentration of at least 200-400 nMantisense oligomer. Typically, one or more doses of antisense oligomerare administered, generally at regular intervals, for a period of aboutone to two weeks. Preferred doses for oral administration are from about1-25 mg oligomer per 70 kg. In some cases, doses of greater than 25 mgoligomer/patient may be necessary. For IV administration, preferreddoses are from about 0.5 mg to 10 mg oligomer per 70 kg. The antisenseoligomer may be administered at regular intervals for a short timeperiod, e.g., daily for two weeks or less. However, in some cases theoligomer is administered intermittently over a longer period of time.Administration may be followed by, or concurrent with, administration ofan antibiotic or other therapeutic treatment. The treatment regimen maybe adjusted (dose, frequency, route, etc.) as indicated, based on theresults of immunoassays, other biochemical tests and physiologicalexamination of the subject under treatment.

C. Monitoring of Treatment

An effective in vivo treatment regimen using the antisenseoligonucleotides of the invention may vary according to the duration,dose, frequency and route of administration, as well as the condition ofthe subject under treatment (i.e., prophylactic administration versusadministration in response to localized or systemic infection).Accordingly, such in vivo therapy will often require monitoring by testsappropriate to the particular type of viral infection under treatment,and corresponding adjustments in the dose or treatment regimen, in orderto achieve an optimal therapeutic outcome. Treatment may be monitored,e.g., by general indicators of infection, such as complete blood count(CBC), nucleic acid detection methods, immunodiagnostic tests, viralculture, or detection of heteroduplex.

The efficacy of an in vivo administered antisense oligomer of theinvention in inhibiting or eliminating the growth of one or more typesof RNA virus may be determined from biological samples (tissue, blood,urine, etc.) taken from a subject prior to, during and subsequent toadministration of the antisense oligomer. Assays of such samples include(1) monitoring the presence or absence of heteroduplex formation withtarget and non-target sequences, using procedures known to those skilledin the art, e.g., an electrophoretic gel mobility assay; (2) monitoringthe amount of viral protein production, as determined by standardtechniques such as ELISA or Western blotting, or (3) measuring theeffect on viral titer, e.g. by the method of Spearman-Karber. (See, forexample, Pari, G. S. et al., Antimicrob. Agents and Chemotherapy39(5):1157-1161, 1995; Anderson, K. P. et al., Antimicrob. Agents andChemotherapy 40(9):2004-2011, 1996, Cottral, G. E. (ed.) in: Manual ofStandard Methods for Veterinary Microbiology, pp. 60-93, 1978).

A preferred method of monitoring the efficacy of the antisense oligomertreatment is by detection of the antisense-RNA heteroduplex. At selectedtime(s) after antisense oligomer administration, a body fluid iscollected for detecting the presence and/or measuring the level ofheteroduplex species in the sample. Typically, the body fluid sample iscollected 3-24 hours after administration, preferably about 6-24 hoursafter administering. As indicated above, the body fluid sample may beurine, saliva, plasma, blood, spinal fluid, or other liquid sample ofbiological origin, and may include cells or cell fragments suspendedtherein, or the liquid medium and its solutes. The amount of samplecollected is typically in the 0.1 to 10 ml range, preferably about 1 mlor less.

The sample may be treated to remove unwanted components and/or to treatthe heteroduplex species in the sample to remove unwanted ssRNA overhangregions, e.g. by treatment with RNase. It is, of course, particularlyimportant to remove overhang where heteroduplex detection relies on sizeseparation, e.g., electrophoresis of mass spectroscopy.

A variety of methods are available for removing unwanted components fromthe sample. For example, since the heteroduplex has a net negativecharge, electrophoretic or ion exchange techniques can be used toseparate the heteroduplex from neutral or positively charged material.The sample may also be contacted with a solid support having asurface-bound antibody or other agent specifically able to bind theheteroduplex. After washing the support to remove unbound material, theheteroduplex can be released in substantially purified form for furtheranalysis, e.g., by electrophoresis, mass spectroscopy or immunoassay.

The following examples illustrate, but are in no way intended to limitthe invention.

Example 1 Antisense Inhibition of West Nile Virus In Vitro

Two PMO oligomers were evaluated for their activity against West Nilevirus in cultured Vero cells. One 20mer PMO oligomer targets the 3′-CSregion of West Nile virus (WNV 3′ CS, SEQ ID NO: 26), the other 20merPMO compound was a “nonsense” sequence (5′-AGTCTCGACTTGCTACCTCA-3′) withno significant homology to any human, monkey or WNV genetic sequence(NC-1). Both PMO oligomers were conjugated at the 5′ end with a peptide(R₉F₂C-5′-PMO) to enhance cellular uptake in vitro. Two separateexperiments, a “two point” and an “eight point dose response”, wereperformed by adding each PMO oligomer, along with virus inoculum, tocells suspended in standard mammalian tissue culture media supplementedwith 2% fetal-calf serum. After 24 hours the cells were scored forcytopathic effect both visually under a microscope, and quantitativelywith a microplate reader using the ‘neutral-red dye assay’ as described(Morrey, Smee et al. 2002). To those skilled in the art, an effectiveconcentration resulting in 50% reduction in viral titer (EC50) of over20 micromolar is considered low antiviral activity while an EC50 under20 micromolar indicates substantial antiviral activity. The tables belowsummarize these results

Two Point Dose Response

PMO compound name EC50 WNV 3′ CS (SEQ ID 26) 7 micromolar NC-1 >20micromolar

Eight Point Dose Response

PMO compound name EC50 WNV 3′ CS (SEQ ID 26) 10 micromolar NegativePMO >56 micromolar

Example 2 Antisense Inhibition of Tick Borne Encephalitis

This example describes a study that was devised to test the antiviralactivity of antisense PMO compounds of the present invention against twoflaviviruses; Tick Borne Encephalitis virus (TBE) and West Nile virus(WN). Two PMO oligomers were evaluated for antiviral activity; TBE 3′CS,SEQ ID NO:25 and; a scramble control sequence DS-scr(5′-AGTCTCGACTTGCTACCTCA-3′). Both PMO oligomers were conjugated at the5′ end with an arginine-rich peptide (R₉F₂C-5′-PMO) to enhance cellularuptake as described (U.S. Patent Application 60/466,703 and Moulton,Nelson et al. 2004). The WN virus infection provided a negative controlinfection as there is no homology between WN and the TBE 3′CS targetingPMO. This control indicates the level of non-specific viral suppressionof each of the PMOs. The PMO compounds were prepared to provide a 2 mMstock solution, which were then titrated against a standard dose ofvirus on tissue culture cells. Cells were infected with a multiplicityof infection (moi) of 1 and the virus yield was assessed in samples ofsupernatant medium taken at 18 hours post infection.

The two virus strains used in this example:

1) TC 401 West Nile 99-34940-31A (New York strain) Passage 2

2) TC 339 Tick Borne Encephalitis virus (Hypr strain) Passage 49

Four T175 tissue culture flasks (NUNC) of SW 13 cells (human caucasianadrenal cortex adenocarcinoma cell line ECAAC 87031801 grown in RPMI1640 medium plus 5% FBS) at passage 130 were washed twice withtrypsin-EDTA (1×) and incubated for 2-3 minutes at 37° C. The cells wereresuspended in 11.5 ml growth medium per flask and pooled. A cell countwas performed on the pooled cell suspension and the result was 1.74×10⁶cells/ml with 99% viability. Six mls of the cell suspension was used toseed four T175 flasks and 40 ml of the cell suspension was diluted to270 ml. This was dispensed in 3 ml aliquots per well in 15 six-wellplates. The plates were incubated overnight to form confluent cellmonolayers.

Each of the PMO compounds was diluted to 25, 20, 15, 10 and 5 μM in 4 mlserum-free RPMI 1640 medium. The medium was removed from the wells oftwo six-well plates. 2 ml of the appropriate compound dilution wasdispensed in all wells of a plate and this was repeated on separateplates for both PMO compounds. The plates were incubated at 37° C. for 5hours. The two viruses were removed from the −70° C. freezer and thawedrapidly. Each virus was diluted to 2×10⁶ pfu/ml to produce 42 mlserum-free medium. The six-well plates were removed from the incubatorand the pre-treatment medium aspirated from all the wells. 1 ml ofmedium was added to each well of the control plate (no compound). Eachset of plates received 1 ml/well of either TBE or WN diluted to 2×10⁶pfu/ml. The plates were incubated at room temperature for 1 hour and themedium was then removed and replaced with 2 ml RPMI 1640 plus 1% FBSplus the same concentration of test compound as used to pre-treat thecells. The plates were incubated at 37° C. for 18 hours.

To prepare 24 well plates for determining virus titers, eight T175tissue culture flasks (NUNC) of SW 13 cells at passage 131 were washedtwice with trypsin-EDTA (1×) and incubated for 2-3 minutes at 37° C. Thecells were resuspended in 11.5 ml growth medium per flask and pooled. Acell count was performed on the pooled cell suspension and the resultwas 1.7×10⁶ cells/ml with 99% viability. 80 ml of the cell suspensionwas diluted to 680 ml. These cells were dispensed as 1 ml per wellaliquots in eight 24-well plates. The plates were incubated overnight toform confluent monolayers.

At 18 hours post-infection the supernatant media from the PMO-treated,virus-infected six-well plates were harvested from each individual well.Thirty μl of each harvest was placed in a single cup of a 96-well platewith 270 μl serum-free medium. The remainder of the sample was placed incryotube and stored at −70° C. The medium was removed from the 24-wellplates and 250 μl of the titration dilutions were transferred from the96-well plates to the 24 well plates which were incubated at 37° C. forone hour. One ml agarose overlay medium was added to each well and afterallowing the agarose to set at room temperature the plates wereincubated at 37° C. for 5 days. After 5 days the plates were removedfrom the incubator, 1 ml 10% Formol saline was added to each well andthe plates were left at room temperature for 3 hours. The plates werewashed under running water to remove the agarose medium and left todrain inverted whilst the remaining plates were washed. Each well thenreceived 1 ml of 0.1% Naphthalene black stain and the plates were leftfor 30 minutes before the stain was removed and the plates washed underrunning water. They were then left to dry (inverted) for 3 hours. Viralplaques were counted to determine the titer.

FIGS. 4A and 4B show the viral titer obtained from the PMO-treatedinfections as % of untreated control, with virus-infected cells infectedwith either TBEV or WNV and treated with either the TBE antisensecompound (FIG. 4A, where the compound has SEQ ID NO:25) or controlcompound (FIG. 4B, scrambled sequence). As seen from a comparison of theviral titers in FIGS. 24A and 24B, there is a reduction in viral titrein all cells (treated and control) with increasing concentrations ofcompound, thought to be due to a cell-toxicity effect of the attachedarginine-rich peptide present in both antisense and control compounds.At a compound concentration of 15 μM and above, there is seen asequence-specific increase in TBE viral inhibition, both relative to WNV(FIG. 24A), and relative to the scrambled-sequence control (comparingFIGS. 24A and 24B).

Example 3 Inhibition of Dengue Virus Serotypes 1-4 with Antisense Pmo

Dengue Fever/Dengue Hemorrhagic Fever (DF/DHF) has become a major globalhealth problem over the past 20 years. Geographic distribution of thedengue virus (DEN), it's mosquito vectors and the disease burden itcauses continue to increase. The World Health Organization estimatesthat there are 50-100 million new infections yearly. DF/DHF is now aleading cause of hospitalization and death among children in southernAsia, and its incidence is sharply rising in the Americas. There iscurrently no vaccine or effective therapeutic. One requirement of asuccessful vaccine or therapeutic is that it be effective against all 4human serotypes of DEN. The purpose of this study was to evaluate theefficacy and specificity of PMO that target the 3′-CS at inhibiting thereplication of four serotypes of DEN in Vero cells in culture. The 5 PMOcompounds were designed to target sequence elements in thepositive-strand DEN2 RNA that have been recognized as important in viraltranscription and/or translation (4,5). The PMO in this study wereconjugated to an arginine-rich peptide in order to facilitate entry intoVero E6 cells.

PMOs designed to hybridize to the 3′-CS region of Dengue 2 virus (DEN2),were evaluated for their ability to inhibit Dengue virus replication inmammalian cell culture. The PMO s were conjugated to a shortarginine-rich peptide (R₉F₂C-5′-PMO) to facilitate their entry intocells in culture. Vero E6 cells were incubated with the PMO agents,inoculated with DEN serotypes 1-4, and viral titer determined byplaque-assay 5-8 days later. The compound targeting 3′-cyclizationsequence (3′CS PMO), reduced the titer of DEN2 by over 3 orders ofmagnitude, compared to controls, in a dose-dependent andsequence-specific manner over a 4-6 day period as shown in FIG. 5A. TenμM solutions of the 3′-CS PMO each reduced the titer of all four Dengueserotypes by over two orders of magnitude, in some cases belowdetectable limits as shown in FIGS. 5B-5D. The effective anti-DENcompounds did not alter the titer of West Nile Virus (WNV) grown in VeroE6 cells. These data indicate that the 3′-CS PMO compound is a potentialDEN 1-4 therapeutic.

From the foregoing, it will be appreciated how various objects andfeatures of the invention are met. Because the target sequences areconserved across several flaviviruses, a single oligonucleotide analogcan be used to treat each of the several viruses. For example, a singleanalog directed against SEQ ID NO:1 or its complement SEQ ID NO:3 may beused for inhibiting replication of St. Louis encephalitis virus, MurrayValley encephalitis virus, West Nile virus, Kunjin virus, Japaneseencephalitis virus, Yellow fever virus, Dengue virus—Types 1, 2, 3, and4, and West Nile virus, and a single analog directed against SEQ ID NO:2 or its complement SEQ ID NO: 4 may be used for treating Tick borneencephalitis virus, Powassen virus, Louping III virus, Kyasanur Forestdisease virus, and Alkhurma virus.

Where the target sequence is limited to one of the complementarycyclization sequences, the analog would be effective in disrupting thecyclization stem secondary structure in both the genomic sense strand,and the first replication, antisense strand, thereby serving to inhibitviral replication at the level of replication of both the positive andnegative strands.

The analog is stable in the body and for some analog structures, e.g.,PMO, may be administered orally. Further, the formation ofheteroduplexes between the analog and viral target may be used toconfirm the presence or absence of infection by a flavivirus, and/or toconfirm uptake of the therapeutic agent by the host.

TABLE 3 Sequence Listing Table SEQ ID NO. Target Sequences5′-UCAAUAUG-3′ 1 5′-GGAGAACAAGA-3′ 2 5′-CAUAUUGA-3′ 3 5′-UCUUGUUCUCC-3′4 5′-GUCAAUAUGCUAAAACGCGG-3′ 5 5′-AUCAAUAUGCUGAAACGCGG-3 65′-GUCAAUAUGGUACGACGAGG-3′ 7 5′-CUUUCAAUAUGCUGAAACGCG-3′ 85′-CUAUCAAUAUGCUGAAACGCG-3′ 9 5′-CAGCUUAGGAGAACAAGAGCUG-3′ 105′-ACAGCAUAUUGACACCUGGGA-3′ 11 5′-UGGGACCAUAUUGACGCCAGGGA-3′ 125-AAACAGCAUAUUGACGCUGGGA-3′ 13 5′-CGGUUCUUGUUCUCCCUGAGCC-3′ 145′-GGCUGUCAAUAUGCUAAAAC-3′ 15 Oligomer Targeting Sequences5′-CCGCGTTTTAGCATATTGAC-3′ 16 5′-CCGCGTTTCAGCATATTGAT-3′ 175′-CCTCGTCGTACCATATTGAC-3′ 18 5′-CGCGTTTCAGCATATTGAAAG-3′ 195-CAGCTCTTGTTCTCCTAAGCTG-3′ 20 5′-GGCTGTCAATATGCTAAAAC-3′ 215′-TCCCAGGTGTCAATATGCTGTT-3′ 22 5′-TCCCTGGCGTCAATATGGTCCCA-3′ 235′-TCCCAGCGTCAATATGCTGTTT-3′ 24 5′-GGCTCAGGGAGAACAAGAACCG-3′ 255′-CAGGTGTCAATATGCTGTTTTG-3′ 26 5′-CCCAGCGTCAATATGCTG-3′ 27

It is claimed:
 1. A method of inhibiting replication of a flavivirus inanimal cells, comprising (a) exposing the cells to an oligonucleotideanalog (i) having a nuclease-resistant backbone, (ii) capable of uptakeby the cells, (iii) containing between 8-40 nucleotide bases, and (iv)having a sequence of at least 8 bases complementary to a region of thevirus' positive strand RNA genome that includes at least a portion ofthe genome's 5′ or 3′ cyclization sequence selected from the groupconsisting of SEQ ID NOS:1-4, and (b) by said exposing, forming withinsaid cells a heteroduplex structure composed of the virus ssRNA and theoligonucleotide, characterized by a T_(m) of dissociation of at least45° C., and having disrupted base pairing between the virus' 5′ and 3′cyclization sequences.
 2. The method of claim 1, wherein theoligonucleotide analog to which the cells are exposed contains asequence that is complementary to at least a portion of the genome's 3′cyclization sequence selected from the group consisting of SEQ ID NOS: 3and
 4. 3. The method of claim 1, wherein the oligonucleotide analog towhich the cells are exposed contains a sequence that is capable offorming a heteroduplex structure with a viral sequence that includes ofa portion of the genome's 5′ cyclization sequence selected from thegroup consisting of SEQ ID NOS:1 and 2 and a complementary portion ofthe genome's 3′ cyclization sequence selected from the group consistingof SEQ ID NOS: 3 and 4, respectively.
 4. The method of claim 1, forinhibiting replication of any of St. Louis encephalitis virus, MurrayValley encephalitis virus, West Nile virus, Kunjin virus, Japaneseencephalitis virus, Yellow fever virus, Dengue virus—Types 1, 2, 3, and4, West Nile virus, wherein the oligonucleotide analog to which thecells are exposed has a sequence of at least 8 bases complementary to aregion of the virus' positive strand RNA genome that includes at least aportion of the genome's 5′ or 3′ cyclization sequence selected from thegroup consisting of SEQ ID NOS:1 and 3, respectively.
 5. The method ofclaim 4, wherein the oligonucleotide analog to which the cells areexposed contains a sequence that is complementary to SEQ ID NO:3.
 6. Themethod of claim 4, wherein the oligonucleotide analog to which the cellsare exposed contains a sequence that is capable of forming aheteroduplex structure with a sequence that includes of a portion of thegenome's 5′ cyclization sequence identified as SEQ ID NO:1 and acomplementary portion of the genome's 3′ cyclization sequence identifiedas SEQ ID NO:
 3. 7. The method of claim 1, for inhibiting replication ofany of Tick borne encephalitis virus, Powassen virus, Louping III virus,Kyasanur Forest disease virus, and Alkhurma virus wherein theoligonucleotide analog to which the cells are exposed has a sequence ofat least 8 bases complementary to a region of the virus' positive strandRNA genome that includes at least a portion of the genome's 5′ or 3′cyclization sequence selected from the group consisting of SEQ ID NOS: 2and 4, respectively.
 8. The method of claim 7, wherein theoligonucleotide analog to which the cells are exposed contains asequence that is complementary to SEQ ID NO:
 4. 9. The method of claim7, wherein the oligonucleotide analog to which the cells are exposedcontains a sequence capable of forming a heteroduplex structure with asequence that includes of a portion of the genome's 5′ cyclizationsequence identified as SEQ ID NO: 2 and the genome's 3′ cyclizationsequence identified as SEQ ID NO:4.
 10. The method of claim 1, whereinthe oligonucleotide analog to which the cells are exposed is composed of8-25 morpholino subunits linked by a substantially uncharged,phosphorous-containing backbone.
 11. The method of claim 1, wherein theoligonucleotide analog to which the cells are exposed is composed ofmorpholino subunits and phosphorus-containing intersubunit linkagesjoining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon ofan adjacent subunit.
 12. The method of claim 11, wherein the morpholinosubunits in the analog to which the cells are exposed are joined byphosphorodiamidate linkages, in accordance with the structure:

where Y₁=O, Z=O, Pj is a purine or pyrimidine base-pairing moietyeffective to bind, by base-specific hydrogen bonding, to a base in apolynucleotide, and X is alkyl, alkoxy, thioalkoxy, amino or alkylamino, including dialkylamino.
 13. The method of claim 12, for treatinga flavivirus infection in a human subject, wherein said exposingincludes administering the oligonucleotide analog orally to the subject.14. The method of claim 12, for treating a flavivirus infection in ahuman subject, wherein said exposing includes administering theoligonucleotide analog to the subject, and which further includesmonitoring a body fluid for the appearance of a heteroduplex composed ofthe oligonucleotide analog and a complementary portion of the viralgenome.
 15. The method of claim 1, for treating West Nile virus in amammalian subject, wherein the oligonucleotide analog to which the cellsare exposed contains the sequence identified as SEQ ID NO:26.
 16. Themethod of claim 1, for treating a Dengue virus, Types 1-4, in amammalian subject, wherein the oligonucleotide analog to which the cellsare exposed contains either of the sequences identified as SEQ ID NOS:24and
 27. 17. An oligonucleotide analog for use in inhibiting replicationof a flavivirus in animal cells, characterized by: (i) anuclease-resistant backbone, (ii) capable of uptake by virus-infectedanimal cells, (iii) containing between 8-40 nucleotide bases, (iv)having a sequence of at least 8 bases complementary to a region of theflavivvirus' positive strand RNA genome that includes at least a portionof the genome's 5′ or 3′ cyclization sequence selected from the groupconsisting of SEQ ID NOS:1-4, and (v) capable of forming with theflavivirus ssRNA genome, a heteroduplex structure characterized by aT_(m) of dissociation of at least 45° C., and having disrupted basepairing between the virus' 5′ and 3′ cyclization sequences.
 18. Theanalog of claim 17, which contains a sequence that is complementary toat least a portion of the viral genome's 3′ cyclization sequenceselected from the group consisting of SEQ ID NOS:3 and
 4. 19. The analogof claim 17, which contains a sequence that is capable of forming aheteroduplex structure with a viral sequence that includes a portion ofthe genome's 5′ cyclization sequence selected from the group consistingof SEQ ID NOS: 1 and 2 and a complementary portion of the genome's 3′cyclization sequence selected from the group consisting of SEQ ID NOS: 3and 4, respectively.
 20. The analog of claim 17, for inhibitingreplication of any of St. Louis encephalitis virus, Murray Valleyencephalitis virus, West Nile virus, Kunjin virus, Japanese encephalitisvirus, Yellow fever virus, Dengue virus—Types 1, 2, 3, and 4, West Nilevirus, which has a sequence of at least 8 bases complementary to aregion of the virus' positive strand RNA genome that includes at least aportion of the genome's 5′ or 3′ cyclization sequence selected from thegroup consisting of SEQ ID NOS:1 and 3, respectively.
 21. The analog ofclaim 20, which contains a sequence that is complementary to SEQ IDNO:3.
 22. The analog of claim 20, which contains a sequence that iscapable of forming a heteroduplex structure with a sequence thatincludes of a portion of the genome's 5′ cyclization sequence identifiedas SEQ ID NO:1 and a complementary portion of the genome's 3′cyclization sequence identified as SEQ ID NO:
 3. 23. The analog of claim17, for inhibiting replication of any of Tick borne encephalitis virus,Powassen virus, Louping III virus, Kyasanur Forest disease virus, andAlkhurma virus, which has a sequence of at least 8 bases complementaryto a region of the virus' positive strand RNA genome that includes atleast a portion of the genome's 5′ or 3′ cyclization sequence selectedfrom the group consisting of SEQ ID NOS: 2 and 4, respectively.
 24. Theanalog of claim 23, which contains a sequence complementary to at leasta portion of SEQ ID NO:
 4. 25. The analog of claim 23, which contains asequence capable of forming a heteroduplex structure with a sequencethat includes of a portion of the genome's 5′ cyclization sequenceidentified as SEQ ID NO: 2 and a complementary portion of the genome's3′ cyclization sequence identified as SEQ ID NO:
 4. 26. The analog ofclaim 17, composed of morpholino subunits and phosphorus-containingintersubunit linkages joining a morpholino nitrogen of one subunit to a5′ exocyclic carbon of an adjacent subunit.
 27. The analog of claim 17,wherein the morpholino subunits are joined by phosphorodiamidatelinkages, in accordance with the structure:

where Y₁=O, Z=O, Pj is a purine or pyrimidine base-pairing moietyeffective to bind, by base-specific hydrogen bonding, to a base in apolynucleotide, and X is alkyl, alkoxy, thioalkoxy, amino or alkylamino, including dialkylamino.
 28. The analog of claim 17, for treatingWest Nile virus, wherein the oligonucleotide analog to which the cellsare exposed contains the sequence identified as SEQ ID NO:26.
 29. Theanalog of claim 17, for use in treating a Dengue virus, Types 1-4,wherein the oligonucleotide analog to which the cells are exposedcontains either of the sequences identified as SEQ ID NOS:24 and 27.